Closed state-coupled C-type inactivation in BK channels

Abstract

Ion channels regulate ion flow by opening and closing their pore gates. K(+) channels commonly possess two pore gates, one at the intracellular end for fast channel activation/deactivation and the other at the selectivity filter for slow C-type inactivation/recovery. The large-conductance calcium-activated potassium (BK) channel lacks a classic intracellular bundle-crossing activation gate and normally show no C-type inactivation. We hypothesized that the BK channel's activation gate may spatially overlap or coexist with the C-type inactivation gate at or near the selectivity filter. We induced C-type inactivation in BK channels and studied the relationship between activation/deactivation and C-type inactivation/recovery. We observed prominent slow C-type inactivation/recovery in BK channels by an extreme low concentration of extracellular K(+) together with a Y294E/K/Q/S or Y279F mutation whose equivalent in Shaker channels (T449E/K/D/Q/S or W434F) caused a greatly accelerated rate of C-type inactivation or constitutive C-inactivation. C-type inactivation in most K(+) channels occurs upon sustained membrane depolarization or channel opening and then recovers during hyperpolarized membrane potentials or channel closure. However, we found that the BK channel C-type inactivation occurred during hyperpolarized membrane potentials or with decreased intracellular calcium ([Ca(2+)]i) and recovered with depolarized membrane potentials or elevated [Ca(2+)]i Constitutively open mutation prevented BK channels from C-type inactivation. We concluded that BK channel C-type inactivation is closed state-dependent and that its extents and rates inversely correlate with channel-open probability. Because C-type inactivation can involve multiple conformational changes at the selectivity filter, we propose that the BK channel's normal closing may represent an early conformational stage of C-type inactivation.

Keywords: BK channel; C-type inactivation; maxi K channel; pore gate; potassium channel.

Fig. 1.

Effects of external K⁺ and mutations at the Y294 position on BK channel slow activation (recovery from inactivated state). (A) Sequence alignment of the pore regions of the BK channel and other channels. The Y294 and Y279 positions are indicated by asterisks. (B–E) Elicited BK channel K⁺ currents in the absence of external K⁺ for WT (B), Y294T (C), and Y294E (D) channels or in the presence of the indicated amounts of external K⁺ for Y294E (E) channels during a long (2-s) membrane depolarization period from a holding potential of 0 mV to 40, 80, 120, and 160 mV. The excised inside-out patches were recorded in bath solution without added Ca²⁺ and chelator. The external K⁺ was fully replaced by NMDG⁺, except in E for which either no NMDG⁺ was used or the residual amount of K⁺ (Inset) was indicated.

Fig. 2.

Effects of voltage and calcium on inactivation in mutant BK channels. (A) Time course of inactivation in Y294E mutant channels at 0 mV in the virtual absence of [Ca²⁺]_i and external K⁺ (replaced by NMDG⁺). The channels were activated by a 2-second depolarization to 180 mV, and then the inactivation was induced by holding the membrane at a certain voltage. A brief (5-ms) membrane depolarization to 180 mV every 2 seconds was used to determine residual available channels for quick activation. (B–E) Time course of inactivation in the virtual absence of external K⁺ (replaced by NMDG⁺) at different holding membrane voltages either in the presence of 85 μM [Ca²⁺]_i for the Y294E (B), Y294K (D), and Y294Q (E) mutant channels or in the virtual absence of [Ca²⁺]_i for the Y294K (C) mutant channel. (F) Plot of the inactivation rates against holding membrane voltages for different Y294 mutant channels at the indicated [Ca²⁺]_i concentrations.

Fig. 3.

Closed-state dependence of inactivation in mutant BK channels. (A) Voltage dependence of BK channel activation for WT and Y294 mutants at 7.3 or 85 μM [Ca²⁺]_i under symmetric K⁺ recording conditions. The gating parameters V_1/2 and z obtained from single Boltzmann function fit at 7.3 μM Ca²⁺ are 17 ± 2 mV and 1.64 ± 0.07 e (n = 4) for WT, 14 ± 2 mV and 1.56 ± 0.15 e (n = 4) for Y294E, 20 ± 3 mV and 1.54 ± 0.14 e (n = 6) for Y294K, and 20 ± 3 mV and 1.70 ± 0.09 e (n = 6) for Y294Q. The V_1/2 and z at 85 μM are −20 ± 2 mV and 1.43 ± 0.19 e (n = 4) for WT and 19 ± 1 mV and 1.66 ± 0.09 e (n = 4) for Y294Q. Error bars represent ±SEM. (B) Relationship between holding membrane voltages and availability of channels for quick activation (noninactivated channels) in Y294Q- and Y294K-mutant channels in the absence and presence of [Ca²⁺]_i. Voltage dependence of channel open probability at 0 and 85 μM [Ca²⁺]_i is included to show state dependence of inactivation. The gray curves are estimated voltage dependence of the channel’s open probabilities at 0 and 85 μM Ca²⁺ based on those measured at the symmetric K⁺ recording condition. (C) Relationship between normalized conductance (G/Gmax) and voltages for Y294E (gray) single-mutant and Y294E/L312N double-mutant (black) channels in the virtual absence of [Ca²⁺]_i. (D) Time courses of available currents upon brief membrane depolarization in Y294E-mutant (gray) and Y294E/L312N-mutant (black) channels. The excised patches were held at −80 mV in the virtual absence of [Ca²⁺]_i.

Fig. 4.

Effects of voltage and calcium concentration on the recovery from inactivation in Y294-mutant channels. Elicited K⁺ currents of Y294E (A and B) and Y294Q (C and D) channels at 0 and 85 μM [Ca²⁺]_i in the absence of external K⁺ upon a 1-s membrane depolarization from a holding potential of −80 mV to 40, 80, 120, and 160 mV. (E) Calculated rates (Tau) of the observed slow recovery processes at different depolarization voltages for Y294Q, -S, -E, and -K mutants at 0 and 85 μM [Ca²⁺]_i by single exponential fits. (F) Relationship between normalized conductance (G/Gmax) and voltages for Y294-mutant channel currents recovered from the inactivated state at the end of 1-s membrane depolarization in the presence of 85 μM [Ca²⁺]_i.

Fig. 5.

Effects of external cations and mutations at the Y279 position on BK channel slow activation (recovery from inactivated state). (A and B) Elicited K⁺ currents of Y279F mutant channels at 0 and 85 μM [Ca²⁺]_i in the absence of external K⁺ (replaced by NMDG⁺) upon 1-s membrane depolarization from a holding potential of −80 mV to 40, 80, 120, and 160 mV. (C) Similar to B, except Na⁺ was used to replace K⁺. (D) Elicited K⁺ currents of Y279W-mutant channels at the same conditions as in A.

Fig. 6.

Schematic state diagrams depicting possible relationships among different channel states of closed (“C”), open (“O”), closed and inactivated (“CI”), and open and inactivated (“OI”). I₁ means an initial step of inactivation.